Abstract P2X receptors are ATP-gated ion channels that are found in the brain. Once activated these channels open a cation selective pore, leading to depolarization and increased neuronal excitability. We have been developing a non invasive approach to track activation of transmitter-gated P2X cation channels. The method exploits the fact that most transmitter-gated cation channels, including P2X receptors, have appreciable calcium fluxes. We engineered P2X receptors to carry calcium sensors near the inner aspect of the pore, and therefore in a nanodomain. We rigorously tested the method for P2X2 receptors. Within a cell, neuron or network this method allows one to image the location of P2X receptors as well as determine when they are activated, with sensitivity equal to whole-cell patch clamp recording. Additionally, the approach is non invasive and provides micrometer scale spatial information. The data show that a FRET based imaging approach can be used as a general method to track the location, regional expression variation, mobility and activation of transmitter-gated P2X channels in neurons, in real time and in living cells. The approach will help reveal when, where and how different receptors are activated during physiological processes. We have two specific aims with which we seek to exploit and refine our new approach. Specific Aim 1: We will employ in vivo expression of the optical reporters to image and identify the regional expression, location and activation of P2X receptors in distinct neurons of the hippocampus from control and epilepsy prone mice. This is because although ATP is known to regulate hyperexcitability associated with epilepsy its precise role is not yet fully understood, largely because there has been no way to measure P2X receptor activation selectively on neuronal processes. Our approach remedies this shortfall and we will use it to image sites of ATP P2X2 receptor activation on neuronal processes from control and epilepsy prone mice. Together with high resolution electrophysiology our approach will allow us to determine precisely how ATP signaling contributes to increased excitability associated with epilepsy. Overall, we will determine the precise roles that ATP signalling plays in the healthy and epileptic hippocampus. Specific Aim 2: We will generate mice expressing optical reporters for P2X2 receptors. These will be invaluable general tools for the ATP signaling community and specifically will shed light on how P2X receptor signaling contributes to epilepsy. We will design and engineer a new generation of P2X constructs that report receptor activation with faster kinetics and higher spatial sensitivity. This will allow us to image fast millisecond time scale P2X receptor mediated signaling in neuronal processes that are inaccessible to electrophysiological methods. PUBLIC HEALTH RELEVANCE: We will study the mechanisms that determine how ATP signaling and P2X2 receptors contribute to a mouse model of epilepsy. In so doing we will establish the basis for understanding the roles of ATP receptors within neuronal networks in general, as well as the specific roles for P2X2 receptors in epilepsy. This is important because the mechanisms that give rise to epilepsy are incompletely understood, and there is an unmet need for its clinical management in humans.